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J. Biol. Chem., Vol. 283, Issue 19, 12877-12887, May 9, 2008

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Sequential Protein Kinase C (PKC)-dependent and PKC-independent Protein Kinase D Catalytic Activation via G_q-coupled Receptors

DIFFERENTIAL REGULATION OF ACTIVATION LOOP SER⁷⁴⁴ AND SER⁷⁴⁸ PHOSPHORYLATION^*

From the Division of Digestive Diseases, Department of Medicine, David Geffen School of Medicine, CURE: Digestive Diseases Research Center and Molecular Biology Institute, UCLA, Los Angeles, California 90095

Protein kinase D (PKD) is a serine/threonine protein kinase rapidly activated by G protein-coupled receptor (GPCR) agonists via a protein kinase C (PKC)-dependent pathway. Recently, PKD has been implicated in the regulation of long term cellular activities, but little is known about the mechanism(s) of sustained PKD activation. Here, we show that cell treatment with the preferential PKC inhibitors GF 109203X or Gö 6983 blocked rapid (1–5-min) PKD activation induced by bombesin stimulation, but this inhibition was greatly diminished at later times of bombesin stimulation (e.g. 45 min). These results imply that GPCR-induced PKD activation is mediated by early PKC-dependent and late PKC-independent mechanisms. Western blot analysis with site-specific antibodies that detect the phosphorylated state of the activation loop residues Ser⁷⁴⁴ and Ser⁷⁴⁸ revealed striking PKC-independent phosphorylation of Ser⁷⁴⁸ as well as Ser⁷⁴⁴ phosphorylation that remained predominantly but not completely PKC-dependent at later times of bombesin or vasopressin stimulation (20–90 min). To determine the mechanisms involved, we examined activation loop phosphorylation in a set of PKD mutants, including kinase-deficient, constitutively activated, and PKD forms in which the activation loop residues were substituted for alanine. Our results show that PKC-dependent phosphorylation of the activation loop Ser⁷⁴⁴ and Ser⁷⁴⁸ is the primary mechanism involved in early phase PKD activation, whereas PKD autophosphorylation on Ser⁷⁴⁸ is a major mechanism contributing to the late phase of PKD activation occurring in cells stimulated by GPCR agonists. The present studies identify a novel mechanism induced by GPCR activation that leads to late, PKC-independent PKD activation.

Received for publication, January 17, 2008 , and in revised form, March 11, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants R0-1 DK 55003, R0-1 DK56930, and P30 DK41301 (to E. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by NIH Grant K22 CA 128883.

² Supported by NIH Grant R21 DK071783.

³ The Ronald S. Hirshberg Chair. To whom correspondence should be addressed: 900 Veteran Ave., Warren Hall, Rm. 11-124, Dept. of Medicine, UCLA School of Medicine. Los Angeles, CA 90095-1786. Tel.: 310-794-6610; Fax: 310-267-2399; E-mail: erozengurt{at}mednet.ucla.edu.

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